Biology subjects

Schuler, B.

Publications and source records attributed to Schuler, B..

13 recordsLinked to original sources

bioRxiv2026

Beyond Equilibrium Ensembles: Time Rescaling in Coarse-Grained Simulations across Single-Molecule and Condensate Regimes

Residue-level coarse-grained simulations provide a powerful route for modeling biomolecular condensates over length and time scales that are difficult to access with atomistic molecular dynamics. Coarse-grained models have been shown to reproduce many aspects of equilibrium phase behavior. However, it remains unclear to what extent such models can reproduce the relative timescales of molecular dynamics. Here, we examine this question for complex coacervates with markedly different dynamics, formed by the highly acidic intrinsically disordered protein prothymosin with four cationic partners: linker histone H1, protamine, polylysine, and polyarginine. Coexistence simulations using a residue-level coarse-grained model reproduce key equilibrium observables from experiments, including dense-phase concentrations, ionic-strength-dependent phase behavior, and chain dimensions in the dense and dilute phases. Dynamics are accelerated in these simulations, but a composition-specific time-rescaling factor captures the ionic-strength dependence of chain reconfiguration times within a given complex coacervate. In contrast, time rescaling is not transferable between dense and dilute phases or across condensate compositions and can depend on the chosen observable. These results show that agreement with measured equilibrium observables does not imply a universally transferable timescale for conformational dynamics in residue-level coarse-grained simulations. However, we find that the required time rescaling strongly correlates with the interaction energy of the protein chains, suggesting that the missing frictional effects arise from protein-protein interactions rather than solely from protein-solvent interactions, reminiscent of internal friction. Our findings highlight the need to combine thermodynamic validation with kinetic calibration when interpreting chain relaxation, molecular diffusion, and material properties from residue-level coarse-grained simulations of biomolecular condensates.

biophysics↗

bioRxiv2026

Predictive all-atom simulations of disordered proteins and biomolecular condensates through osmometry-guided force-field optimization

All-atom simulations with explicit solvent can provide a detailed and accurate description of dynamics and mechanisms in biomolecular systems, including intrinsically disordered proteins (IDPs) and their condensates. However, interactions involving charged residues and ions remain a persistent source of systematic error. Here we introduce an osmometry-guided optimization strategy that directly targets residue-residue, residue-ion and ion-ion interactions. Osmotic pressure provides key experimental information on molecular interactions and can be calculated directly and rapidly from simulations, enabling iterative force-field optimization. The resulting parameters improve agreement with single-molecule FRET data for IDPs, NMR relaxation data for an IDP-folded-domain complex, and chain dynamics and dimensions in biomolecular condensates of charged IDPs. For such condensates, simulations with our osmometry-optimized force field provide the missing link for predicting condensate dynamics across length and time scales. The strategy is broadly extensible to other interaction classes, including those governing protein-nucleic-acid assemblies.

biophysics↗

bioRxiv2026

Cooperativity, dynamics, and the free-energy surfaces of charge-patterned IDPs

The free-energy surfaces that underlie the conformational distributions of intrinsically disordered proteins (IDPs) are shallow and lack the deep minima characteristic of stable, folded structures. However, even in the absence of secondary or tertiary structure, sequence patterning can lead to conformational preferences and changes in chain dimensions as a function of solution conditions. While patterning effects have received extensive attention from simulation and theory, there is little corresponding data from experiment. Here we investigate the impact of charge patterning on chain dimensions and dynamics in a set of specifically designed polyampholytic IDP variants across the natural range of charge segregation with single-molecule FRET, nanosecond fluorescence correlation, circular dichroism, and NMR spectroscopy. We find that the conformational ensembles and their cooperative response to salt concentration show prominent and systematic dependencies on charge patterning, and to some extent on residue type. In contrast, the chain dynamics remain in the tens-of-nanosecond range, consistent with the absence of pronounced free-energy barriers. In close combination with molecular simulations, we show how the concept of susceptibility can be used to quantify cooperativity in the absence of barriers and relate it to the shallow free-energy surfaces of IDPs.

biophysics↗

bioRxiv2026

Phosphorylation tunes electrostatically driven protein-RNA interactions

Phosphorylation of intrinsically disordered proteins (IDPs) is essential for regulating biomolecular interactions in many cellular processes. However, a quantitative understanding of how phosphorylation tunes the affinity between highly charged IDPs and nucleic acids is lacking. Here, we show that multi-site phosphorylation of the disordered arginine/serine-rich (RS) domain of the splicing factor SRSF1 acts as an electrostatic rheostat that governs RNA binding. By combining enzymatic phosphorylation, phosphomimetic variants, and chemically synthesised phosphopeptides with single-molecule Forster resonance energy transfer measurements, we reveal the RS domain to be a potent driver of protein-RNA association. Increasing phosphorylation progressively reduces this interaction, and extensive phosphorylation eliminates detectable RNA binding. Remarkably, the binding free energy depends linearly on RS-domain net charge, regardless of whether the charge arises from phosphorylation or acidic residues introduced as phosphomimetics. Together, our findings uncover a quantitative framework for how phosphorylation tunes the interactions of charged IDPs and rationalize why two acidic residues are required to mimic a single phosphorylation event.

biophysics↗

bioRxiv2026

Time-Resolved Single-Molecule FRET Reveals Length-Dependent Nucleosome Decompaction by Poly(ADP-ribose)

Highly charged chains of poly(ADP-ribose) (PAR) are synthesized in the cell as part of their central role in DNA damage response. However, the effects of PAR on nucleosome structure and dynamics remain incompletely understood. Here we combine droplet-based microfluidic mixing with single-molecule Forster resonance energy transfer spectroscopy to resolve the kinetics of PAR-induced nucleosome decompaction in non-equilibrium measurements with millisecond time resolution. This approach avoids surface-adhesion and enables the tether-free observation of nucleosome remodeling. We find that PAR triggers nucleosome decompaction via a length-dependent kinetic threshold: Chains with less than ten ADP-ribose units act slowly and weakly, whereas longer PAR polymers induce efficient and rapid nucleosome opening. The extent and reversibility of decompaction further depend on PAR concentration and ionic strength, reflecting a mechanism dominated by electrostatic interactions. Enzymatic PAR digestion demonstrates that PAR can promote both reversible linker DNA opening and irreversible nucleosome disassembly. Coarse-grained molecular simulations suggest that these effects arise from a competition between PAR and DNA for histone tail binding. Altogether, our results establish PAR length as a key factor controlling chromatin accessibility during DNA repair and highlight droplet-based microfluidics as a powerful platform for studying such biomolecular interactions.

biophysics↗

bioRxiv2025

Dynamical buffering of reconfiguration dynamics in intrinsically disordered proteins

The dynamics of intrinsically disordered proteins are important for their function, allowing their heterogeneous conformational ensembles to rapidly reconfigure in response to binding partners or changes in solution conditions. However, the relation between sequence composition and chain dynamics has rarely been studied. Here, we characterize the dynamics of a set of 16 naturally occurring disordered regions of identical chain length but with highly diverse sequences. In spite of the strong variation of chain dimensions with sequence in this set inferred from single-molecule FRET, nanosecond fluorescence correlation spectroscopy yields chain reconfiguration times that are almost independent of sequence. This surprising observation contrasts with the slowdown in dynamics, attributed to internal friction, that has been observed in more compact disordered proteins. We investigated this effect with the aid of multi-microsecond, all-atom explicit-solvent simulations of all 16 disordered proteins. The simulations reproduce the experimental FRET efficiencies with near-quantitative accuracy, with explicit inclusion of the FRET dyes improving agreement with experiment while minimally perturbing the protein ensemble. Critically, the simulations also reproduce the lack of correlation between reconfiguration times and chain dimensions across the sequences and allow us to rationalize this observation as arising from two competing factors as the chains get more compact. The narrowing of end-to-end distance distributions and a concomitant reduction of the corresponding intrachain diffusion coefficients have opposite effects that end up resulting in only a small variation of reconfiguration times with chain dimensions. These compensating factors "buffer" the effect of sequence on linker dynamics, which may help to conserve function as sequences evolve.

biophysics↗

bioRxiv2024

The molecular basis of lamin-specific chromatin interactions

In the cell nucleus, chromatin is anchored to the nuclear lamina, a network of lamin filaments and binding proteins that underly the inner nuclear membrane. The nuclear lamina is involved in chromatin organisation through the interaction of lamina-associated domains (LADs) within the densely packed heterochromatin regions. Employing cryo-focused ion beam (cryo-FIB) milling in conjunction with cryo-electron tomography (cryo-ET), we analysed the distribution of nucleosomes at the lamin-chromatin interface. Depletion of lamin A/C reduced the concentration of nucleosomes at the nuclear periphery, suggesting that lamins are directly involved in the interaction with chromatin. Using cryo-electron microscopy (cryo-EM), we then identified the specific binding motif of the lamin A tail domain that interacts with nucleosomes, distinguishing it from the other lamin isoforms. Furthermore, we examined chromatin structure dynamics using a genome-wide analysis that revealed lamin-dependent macroscopic-scale alterations in gene expression and chromatin remodelling. Our findings provide detailed insights into the dynamic and structural interplay between lamin isoforms and chromatin, molecular interactions which are shaping chromatin architecture and epigenetic regulation.

cell biology↗

bioRxiv2024

The importance of stereochemistry in the disorder-order continuum of protein-protein interactions

Intrinsically disordered proteins can bind via the formation of highly disordered protein complexes without the formation of 3D-structure. Most naturally occurring proteins are "left-handed" or levorotatory (L), made up only of L-amino acids, imprinting molecular structure and communication with stereochemistry. In contrast, their mirror image "right-handed" or dextrorotatory (D) amino acids are rare in Nature. Whether disordered protein complexes are truly independent of 3D-topology and thus of chiral constraints is not clear. To test the chiral constraints of disordered protein-protein interactions, a set of interacting protein pairs covering the disorder-order continuum was chosen as representative examples. By observing both the natural ligands and their stereochemical mirror images in free and bound states, we discovered that chirality was inconsequential in a fully disordered complex. However, if the interaction relied on the ligand undergoing coupled folding and binding, correct stereochemistry was essential. Between these extremes, binding could be observed for the D-ligand with a strength that correlated with the amount of disorder in the final complex. These findings have important implications for our understanding of protein-protein interactions, the molecular processes leading to complex formation, the use of D-peptides in drug discovery, and the chemistry of protein evolution of the first living entities on Earth.

biophysics↗

bioRxiv2023

Single-Molecule FRET at 10 MHz Count Rates

A bottleneck in many studies utilizing single-molecule Forster Resonance Energy Transfer (smFRET) is the attainable photon count rate as it determines the temporal resolution of the experiment. As many biologically relevant processes occur on timescales that are hardly accessible with currently achievable photon count rates, there has been considerable effort to find strategies to increase the stability and brightness of fluorescent dyes. Here, we use DNA nanoantennas to drastically increase the achievable photon count rates and to observe fast biomolecular dynamics in the small volume between two plasmonic nanoparticles. As a proof of concept, we observe the coupled folding and binding of two intrinsically disordered proteins which form transient encounter complexes with lifetimes on the order of 100 s. To test the limits of our approach, we also investigated the hybridization of a short single-stranded DNA to its complementary counterpart, revealing a transition path time of 17 s at photon count rates of around 10 MHz, which is an order-of-magnitude improvement when compared to the state of the art. Concomitantly, the photostability was increased, enabling many seconds long megahertz fluorescence time traces. Due to the modular nature of the DNA origami method, this platform can be adapted to a broad range of biomolecules, providing a promising approach to study previously unobservable ultrafast biophysical processes.

biophysics↗

bioRxiv2023

A quantitative description for optical mass measurement of single biomolecules

Label-free detection of single biomolecules in solution has been achieved using a variety of experimental approaches over the past decade. Yet, our understanding of the magnitude of the optical contrast and its relationship to the underlying atomic structure, as well as the achievable measurement sensitivity and precision remain poorly defined. Here, we use a Fourier optics approach combined with an atomic structure-based molecular polarizability model to simulate mass photometry experiments from first principles. We find excellent agreement between several key experimentally-determined parameters such as optical contrast-to-mass conversion, achievable mass accuracy and molecular shape/orientation dependence. This allows us to determine detection sensitivity and measurement precision that is mostly independent of the optical detection approach chosen, resulting in a general framework for light-based single molecule detection and quantification.

biophysics↗

bioRxiv2022

Ultrafast molecular dynamics observed within a dense protein condensate

Many biological macromolecules can phase-separate in the cell and form highly concentrated condensates. The mesoscopic dynamics of these assemblies have been widely characterized, but their behavior at the molecular scale has remained more elusive. Here we investigate condensates of two highly charged disordered human proteins as a characteristic example of liquid-liquid phase separation. The dense phase is 1000 times more concentrated and has 300 times higher bulk viscosity than the dilute phase. However, single-molecule spectroscopy in individual droplets reveals that the polypeptide chains are remarkably dynamic, with sub-microsecond reconfiguration times. We rationalize this behavior with large-scale all-atom molecular-dynamics simulations, which reveal an unexpectedly similar short-range molecular environment in the dense and dilute phases, suggesting that local biochemical processes and interactions can remain exceedingly rapid in phase-separated systems.

biophysics↗

bioRxiv2021

Global Structure of the Intrinsically Disordered Protein Tau Emerges from its Local Structure

The paradigmatic disordered protein tau plays an important role in neuronal function and neurodegenerative diseases. To disentangle the factors controlling the balance between functional and disease-associated conformational states, we build a structural ensemble of the tau K18 fragment containing the four pseudorepeat domains involved in both microtubule binding and amyloid fibril formation. We assemble 129-residue-long tau K18 chains at atomic resolution from an extensive fragment library constructed with molecular dynamics simulations. We introduce a reweighted hierarchical chain growth (RHCG) algorithm that integrates experimental data reporting on the local structure into the assembly process in a systematic manner. By combining Bayesian ensemble refinement with importance sampling, we obtain well-defined ensembles and overcome the problem of exponentially varying weights in the integrative modeling of long-chain polymeric molecules. The resulting tau K18 ensembles capture nuclear magnetic resonance (NMR) chemical shift and J-coupling measurements. Without further fitting, we achieve excellent agreement with measurements of NMR residual dipolar couplings. The good agreement with experimental measures of global structures such as singlemolecule Forster resonance energy transfer (FRET) efficiencies is improved further by ensemble refinement. By comparing wild-type and mutant ensembles, we show that pathogenic single-point P301 mutations shift the population from the turn-like conformations of the functional microtubule-bound state to the extended conformations of disease-associated tau fibrils. RHCG thus provides us with an atomically resolved view of the population equilibrium between functional and aggregation-prone states of tau K18, and demonstrates that global structural characteristics of this intrinsically disordered protein emerge from its local structure.

biophysics↗

bioRxiv2020

Disordered Proteins Enable Histone Chaperoning on the Nucleosome

Proteins with highly charged disordered regions are abundant in the nucleus, where many of them interact with nucleic acids and control key processes such as transcription. The functional advantages conferred by protein disorder, however, have largely remained unclear. Here we show that disorder can facilitate a remarkable regulatory mechanism involving molecular competition. Single-molecule experiments demonstrate that the human linker histone H1 binds to the nucleosome with ultra-high affinity. However, the large-amplitude dynamics of the positively charged disordered regions of H1 persist on the nucleosome and facilitate the interaction with the highly negatively charged and disordered histone chaperone prothymosin . Consequently, prothymosin can efficiently invade the H1-nucleosome complex and displace H1 via competitive substitution. By integrating experiments and simulations, we establish a molecular model that rationalizes this process structurally and kinetically. Given the abundance of charged disordered regions in the nuclear proteome, this mechanism may be widespread in cellular regulation.

biophysics↗